Growth is the process through which cells accumulate mass and increase in size. mTORC1 is a protein kinase composed of the mTOR catalytic subunit and the associated proteins raptor and mLST8 and the central component of a signaling network that regulates growth in response to growth factors, nutrients, and stress. It is increasingly apparent that many cancer-promoting lesions activate the mTORC1 pathway. Most notably, the TSC1-TSC2 tumor suppressor complex--whose inactivation causes the tumor- prone syndrome Tuberous Sclerosis Complex (TSC) and the related disease Lymphangioleiomyomatosis (LAM)--is a major negative regulator of mTORC1. The TSC1-TSC2 heterodimer is a GTPase activating protein (GAP) that inhibits rheb, a GTP-binding protein that activates mTORC1 through a poorly understood mechanism. TSC1-TSC2 and rheb are also important for the activation of mTORC1 that occurs in cells that have lost the PTEN, NF1, LKB1, or p53 tumor suppressors. We propose to address key gaps in our understanding of mTORC1 biology. First, we will determine the molecular mechanisms that activate mTORC1 in response to growth factors or inactivation of TSC1- TSC2 or PTEN. Second, using mouse models we are developing, we will rigorously test the role of mTORC1 in tumorigenesis caused by inactivation of PTEN. Third, we will obtain structural information about intact mTORC1 and the mTOR kinase domain. We will accomplish our goals with a collaborative multi-disciplinary approach that exploits the tools of biochemistry, molecular biology, mouse models of cancer, and structural biology. We believe that our results are likely to have significant medical implications for the treatment of Tuberous Sclerosis Complex. An understanding of how the inactivation of TSC1-TSC2 activates mTORC1 is necessary for the rational development of therapies for TSC. With our animal models we will obtain a definitive genetic answer to the potential value of inhibiting mTORC1 in patients with tumors missing PTEN. Lastly, modified versions of our novel mTORC1 kinase assay may be useful for the high- throughput screening of small molecules that inhibit mTORC1 and our structural work will inform the development of inhibitors of the mTOR kinase. PUBLIC HEALTH RELEVANCE: Growth is the process through which cells and organisms accumulate mass and increase in size. It is increasingly apparent that this basic biological process is deregulated in common human diseases, most notably in cancer. In this application we propose to study one of the major growth regulators in mammals, a complex of several proteins called mTORC1. We propose to elucidate the molecular mechanisms that activate mTORC1 in cancer and normal cells, to determine if inhibiting mTORC1 is likely to be a good treatment for tumors that have a common cancer-causing genetic alteration, and, lastly, to determine the molecular structure of mTORC1. The overall goal of our proposed work is to increase the capacity of the oncology community to rationally exploit mTORC1 in the treatment of cancer. We anticipate that our work will help understand which tumor classes should be treated with mTORC1 inhibitors, aid in the development of more specific mTORC1 inhibitors, and lead to the discovery of mechanisms that may be targets for future drug development.